PROJECT SUMMARY The obesity pandemic is accelerating the risk of diabetes, non-alcoholic fatty liver disease (NAFLD), cardio- vascular diseases and other ailments. PPARa agonist drugs are prescribed to promote fat loss and amelio- rate hepatic steatosis and insulin resistance by activating gene programs driving mitochondrial b-oxidation. Effects of PPARa agonists in the liver are enhanced by remodeling the action of MAMs, cytoplasmic struc- tures that foster exchange of Ca2+ and phospholipids between the mitochondrion and endoplasmic reticu- lum. The master mTORC2/Akt signaling node localizes to the MAM where it coordinates PPARa and MAM actions through an unknown mechanism. Therefore, understanding how mTORC2/Akt regulates PPARa and MAMs in the liver is crucial to developing new approaches to combat the obesity epidemic. Our long- term goal is to understand how nuclear gene expression and cytoplasmic ER/mitochondrial activity are coor- dinated to control energy homeostasis in humans. The objective of this particular application is to determine how mTORC2/Akt-mediated phosphorylation of the multi-functional protein PACS-2 Ser437 combines with PACS-2 nuclear trafficking signals to coordinate PPARa transcriptional activity with MAM-dependent calci- um exchange in response to fasting or a high fat diet. Our central hypothesis is that in response to overeat- ing, mTORC2/Akt-phosphorylated PACS-2 sequesters PPARa in the cytoplasm and increases MAMs, there- by repressing genes controlling fatty acid oxidation while inducing calcium overload in mitochondria. These combined effects cause steatosis and insulin resistance. By contrast, fasting silences mTORC2/Akt signal- ing, triggering PACS-2 dephosphorylation. Consequently, PPARa is liberated and MAMs are remodeled, which combine to increase fatty acid oxidation and support fasting-induced autophagy. Guided by strong preliminary data, we will test our hypothesis by pursuing three specific aims: 1) Determine how liver PACS- 2 coordinates PPARa-dependent gene expression with MAM remodeling to regulate fatty acid oxidation; 2) Determine how mTOR/Akt controls PACS-2 regulation of PGC-1a/PPARa activity; and 3) Determine how PACS-2 regulates access of PPARa to the nucleus. The approach is innovative because it will combine in vivo models of overnutrition and fasting together with studies on gene expression, ER-mitochondria commu- nication, mitochondrial oxygen consumption and live-cell imaging to describe a novel and previously unrec- ognized pathway controlling the response to fasting or a high fat diet. This research is significant because it will advance our understanding of how mTORC2/Akt controls PACS-2 Ser437 to act as a molecular switch to coordinate vital, homeostatic transcriptional and mitochondrial responses to nutrient stresses.